EN 14133:2003

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Ochratoxin A in Wein und Bier - HPLC-Verfahren mit Reinigung an einer ImmunoaffinitätssäuleProduits alimentaires - Dosage de l'ochratoxine A dans le vin et la biere - Méthode par CLHP apres purification sur colonne d'immunoaffinitéFoodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up67.160.10Alcoholic beveragesICS:Ta slovenski standard je istoveten z:EN 14133:2003SIST EN 14133:2003en01-november-2003SIST EN 14133:2003SLOVENSKI
STANDARD



SIST EN 14133:2003



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14133July 2003ICS 67.160.10English versionFoodstuffs - Determination of ochratoxin A in wine and beer -HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage de l'ochratoxine A présentedans le vin et la bière - Méthode par CLHP et parpurification en colonne d'immunoafinitéLebensmittel - Bestimmung von Ochratoxin A in Wein undBier - HPLC-Verfahren mit Reinigung an einerImmunoaffinitätssäuleThis European Standard was approved by CEN on 16 May 2003.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14133:2003 ESIST EN 14133:2003



EN 14133:2003 (E)2ContentsForeword.31Scope.32Normative references.33Principle.34Reagents.45Apparatus.56Procedure.67HPLC analysis.78Calculation.89Precision.810Test report.9Annex A (informative)
Precision data.11Bibliography.13SIST EN 14133:2003



EN 14133:2003 (E)3ForewordThis document (EN 14133:2003) has been prepared by Technical Committee CEN/TC 275 "Food analysis -Horizontal methods", the secretariat of which is held by DIN.This
European Standard shall be given the status of a national standard, either by publication of an identical text orby endorsement, at the latest by January 2004, and conflicting national standards shall be withdrawn at the latestby January 2004.Annex A is informative.WARNING — Ochratoxin A is a potent nephrotoxin and liver toxin and has been reported to haveimmunosuppressant properties. It is classified by the International Agency for Research on Cancer (IARC)as possibly carcinogenic to humans (Group 2B). Acetonitrile is hazardous. Toluene is highly flammableand harmful. Observe appropriate safety precautions for handling such compounds. Gloves and safetyglasses shall be worn at all times and all standard and sample preparation stages shall be carried out in afume cupboard. Operation outside the fume cupboard, such as measurement of standards by UVspectrometer, shall be performed with the standard in closed containers. Decontamination procedures forlaboratory wastes have been reported by the International Agency for Research on Cancer (IARC), see [1].According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal,Slovakia, Spain, Sweden, Switzerland and the United Kingdom.1 ScopeThis European Standard specifies a method for the determination of ochratoxin A content in wine and beer usingimmunoaffinity column clean up and high performance liquid chromatography (HPLC), see [2], [3].This method has been validated in an interlaboratory study according to AOAC International Guidelines [4] forcollaborative study procedures to validate characteristics of a method of analysis for the determination ofochratoxin A in wine and beer via the analysis of naturally contaminated and spiked samples of wine and beer atlevels ranging from 0,1 ng/ml to 3 ng/ml.2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate places in the text and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).EN ISO 3696:1995, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987).3 PrincipleWine and beer samples are diluted with a solution containing polyethylene glycol (PEG) and sodium hydrogencarbonate, filtered and cleaned up by immunoaffinity column. Ochratoxin A is eluted with methanol and quantifiedby reversed-phase HPLC with fluorescence detection.NOTEThe use of PEG is essential to increase ochratoxin A recoveries and to reduce the number and intensity of otherchromatographic peaks.SIST EN 14133:2003



EN 14133:2003 (E)44 Reagents4.1 GeneralUnless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water ofgrade 1 as defined in EN ISO 3696:1995.Commercially available reagents with equivalent properties to the ones listed may be used.4.2 Sodium chloride4.3 Sodium hydrogen carbonate1.4 Polyethylene glycol, (average molecular weight of 8000)4.5 Methanol, HPLC grade4.6 Acetonitrile, HPLC grade4.7 Water, HPLC grade4.8 Glacial acetic acid, (CH3COOH)
%4.9 Diluting solutionDissolve 10 g polyethylene glycol (4.4) and 50 g sodium hydrogen carbonate (4.3) in approximately 950 ml waterand bring up to 1000 ml with water.4.10 Washing solutionDissolve 25 g sodium chloride (4.2) and 5 g sodium hydrogen carbonate (4.3) in approximately 950 ml water andbring up to 1000 ml with water.4.11 HPLC mobile phaseMix 990 ml HPLC water (4.7) with 990 ml acetonitrile (4.6) and 20 ml glacial acetic acid (4.8), filter through 0,45 µmfilter and degas (e.g. with helium).4.12 Toluene4.13 Solvent mixture of toluene and glacial acetic acidMix 99 parts per volume of toluene (4.12) with 1 part per volume of glacial acetic acid (4.8).4.14 Ochratoxin A stock solutionDissolve 1 mg of ochratoxin A (in crystal form) or the contents of 1 ampoule (if ochratoxin A has been obtained as afilm) in solvent mixture (4.13) to give a solution containing approximately 20 µg/ml to 30 µg/ml of ochratoxin A. Todetermine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in5 nm steps in 1 cm quartz cells (5.10) and solvent mixture (4.13) as reference. Identify the wavelength formaximum absorption and calculate the mass concentration of ochratoxin A, OTA, in micrograms per millilitre, usingequation (1):der´´´=1000AmaxotaM(1)SIST EN 14133:2003



EN 14133:2003 (E)5where:Amax is the absorption determined at the maximum of the absorption curve (here: at 333 nm);M is the relative molecular mass of ochratoxin A (M = 403,8 g/mol);e is the relative molar absorption coefficient of ochratoxin A in solvent mixture (4.13)(here: e = 5440 m2/mol);d is the path length of the quartz cell in centimetres.This solution is stable at –18 °C for at least 4 years.4.15 Ochratoxin A standard solutionDilute the stock solution (4.14) with solvent mixture (4.13) to obtain a standard solution with a mass concentrationof ochratoxin A of 2 µg/ml. Store this solution in a refrigerator at approximately 4 °C and check the stability.4.16 Ochratoxin A calibration solutionPipette 0,5 ml of the 2 µg/ml ochratoxin A standard solution (4.15) into a 10 ml volumetric flask (5.3) and evaporatethe solvent under a stream of nitrogen. Redissolve in 10 ml of mobile phase (4.11). This gives a massconcentration of 100 ng/ml ochratoxin A.Prepare six HPLC calibrants in separate 5 ml volumetric flasks (5.3) according to Table 1. Dilute each solution to5 ml with HPLC mobile phase (4.11).Table 1 — Preparation of calibration solutionsStd 1Std 2Std 3Std 4Std 5Std 6µl of filtered mobile phase (4.11)497049004700400030002000µl of 100 ng/ml OTA solution30100300100020003000OTA mass concentration (ng/ml)0,62,06,0204060injected ng OTA0,060,200,602,004,006,00Prepare the calibration solutions at the beginning of every day of the analysis.4.17 Immunoaffinity columnsThe immunoaffinity column shall contain antibodies raised against ochratoxin A. The column shall have a totalbinding capacity of not less than 100 ng of ochratoxin A and shall give a recovery of not less than 85 % when adiluted wine solution containing 100 ng of ochratoxin A is applied.5 ApparatusUsual laboratory equipment and, in particular, the following:5.1 Microbalance, capable to measure 0,01 mg5.2 Glass vials, approximately 4 ml, with polytetrafluoroethylene (PTFE)-lined screw cap, or appropriate sealablescrew cap.Certain types of vials can lead to losses of ochratoxin A during evaporation. To avoid this, silanization can beapplied. Prepare vials by filling them with silanizing reagent and leave this reagent in the vial for one minute. Rinsethe vial twice with appropriate solvent (toluene, acetone or hexane) followed by water (twice) and dry the vial.SIST EN 14133:2003



EN 14133:2003 (E)65.3 Volumetric flasks, 5 ml and 10 ml volume5.4 Vacuum manifold, to accommodate immunoaffinity columns5.5 Reservoirs and attachments, to fit to immunoaffinity columns5.6 Glass microfibre filters, pore size 1,6 µm, (e.g. Whatman GF/A1) or equivale
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